Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Abstract

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis

“…Extracting POSs from bovine eyes is a crucial step in experimental procedures. However, this process presents inherent challenges due to their sensitivity and fragility, their small size, and their deep retinal location [ 28 ]. Studies have reported the delicate nature of POSs, which are prone to damage and disruption during extraction [ 28 ].…”

“…However, this process presents inherent challenges due to their sensitivity and fragility, their small size, and their deep retinal location [ 28 ]. Studies have reported the delicate nature of POSs, which are prone to damage and disruption during extraction [ 28 ]. Temperature changes, light exposure, and mechanical stress have been identified as factors that can affect the structural and functional integrity of POSs [ 27 , 29 ].…”

Laminin-defined Mechanical Status Modulates Retinal Pigment Epithelium Functionality

“…Photoreceptor outer segment internalisation assay. Photoreceptor outer segments (POS) were isolated in batches from porcine eyes from a local slaughterhouse as previously described (Parinot et al, 2014).…”

“…Photoreceptor outer segments (POS) were isolated in batches from porcine eyes from a local slaughterhouse as previously described (Parinot et al , 2014). Briefly, the eyes were dissected using a red safelight lamp as the only illumination source, and the neural retina was collected.…”